Abstract CFTR?s complex folding scheme leads to near-complete ER-associated degradation (ERAD) of the common CF disease mutant, F508del CFTR, and of a significant fraction of the WT protein. Defects in protein folding and aggregation underlie a diverse set of ~100 diseases, and their treatment requires an understanding of the checkpoints that determine protein fate. We previously identified novel physical and functional interactions of CFTR with the small heat shock protein (sHsp), Hsp27, which led to mutant CFTR conjugation with the small ubiquitin-like modifier, SUMO. F508del CFTR was selectively degraded by this Hsp27/SUMO pathway, by involving a SUMO-targeted ubiquitin ligase, RNF4, to target mutant CFTR linked to SUMO-2/3 poly-chains. These findings identified the first nonnuclear pathway for SUMO modification and degradation of an integral membrane protein. Protoarray analysis for SUMO binding proteins identified the SUMO E3 enzyme, PIAS 4, which modifies CFTR with SUMO-1, a paralog that cannot form poly-chains, and therefore obviates RNF4-mediated degradation. PIAS4 stabilizes the immature forms of WT, F508del and numerous rarer CFTR misfolding variants, and it increased the efficacy of correctors of F508del CFTR trafficking to the plasma membrane. With this Preliminary Data, the current proposal will evaluate the hypothesis that different SUMO paralogs mediate CFTR biogenesis vs. degradation using different components of the SUMOylation pathway. Aim 1 focuses on the mechanisms of PIAS4/SUMO-1 induced CFTR biogenesis. It asks whether and how SUMO-1 modification stabilizes F508del, and it relies on purification of WT CFTR and its NBD subdomains, as well as limited proteolysis to assess the mechanistic basis of PIAS4-induced stability. This aim examines the mechanism of the SUMO paralog switch determines CFTR fate: biogenesis vs. degradation. Aim 2 explores the generality of the ability of PIAS4/SUMO-1 to enhance corrector action for numerous rarer folding mutants, which has allowed their partitioning into three classes of corrector response. Selected variants from these groups will be examined to ask whether their behavior correlates with inherent differences in stability and protease sensitivity. Aim 3 uses results from the SUMO Protoarray to identify HDAC6 as a mediator of non- proteasomal degradation of specific mutants with the hypothesis that chaperone-mediated autophagy is required for their disposal, and it will evaluate the ability of current small molecules to provide therapy of these variants. It is critical that these studies of CFTR fate are performed in airway cells and wherever possible, in differentiated primary cultures of human bronchial epithelia (HBE), whose phenotype has been predictive of small molecule efficacy in clinical studies. This project will provide a mechanistic understanding of new quality control pathways and define their impact on the conflict between protein folding and degradation.